The proposed research will utilize selected aromatase inhibitors as biochemical tools in studying the active site of aromatase. Investigations by the P.I. have demonstrated enhanced affinity of microsomal aromatase for several 7 alpha-thioether substituted 4- androstene-3,17-diones, particularly analogs with aryl amine functionalities. New 7-substituted C19-steroids with enhanced affinity for the enzyme and/or greater metabolic stability have recently been developed and will be used to examine the aromatase protein. Specifically: (1) Aromatase will be purified from human placental microsomes by cholate solubilization, ammonium sulfate fractionation, and a combination of affinity and adsorption chromatography. (2) Radiolabeled 3H-and 125I- analogs of selected enzyme-activated inhibitors will be prepared. New synthetic compounds will be examined in microsomal preparations for inhibitory activity. (3) The radiolabeled enzyme- activated inhibitors prepared above will be incubated with purified aromatase preparations to examine the extent of irreversible binding. Inhibitor-bound aromatase protein will be isolated and treated with proteolytic enzymes and peptides will be separated by HPLC. The amino acid sequence of radiolabeled peptide fragments will be determined by microsequence analysis. Thus, radiolabeled analogs of selected inhibitors will be prepared and utilized to probe the active site of purified aromatase. Knowledge of the chemistry of the enzymatic "pocket" of aromatase is critical in the further design and development of more effective and potent agents for the control of estrogenic processes and for the treatment of advanced estrogen-dependent breast cancer.